Int. J. Pharm. Sci. Rev. Res., 37(2), March – April 2016; Article No. 27, Pages: 151-155
ISSN 0976 – 044X
Research Article
Micellar Liquid Chromatographic Method for Simultaneous Determination of Atenolol and
Aspirin in Bulk and Pharmaceutical Dosage Form
Savita S. Yadav*, Janhavi R.Rao
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Pune, India.
*Corresponding author’s E-mail: savitang2729@yahoo.com
Accepted on: 06-03-2016; Finalized on: 31-03-2016.
ABSTRACT
Micellar liquid chromatographic method has been developed for the simultaneous determination of Atenolol and Aspirin in a bulk
drug and pharmaceutical formulation. Chromatographic separation achieved isocratically on ODS hypersil C18 column (5 μm, 250
mm × 4.6 mm) and micellar mobile phase of 0.07M sodium dodecyl sulfate (SDS) pH 3 adjusted with phosphate buffer and 15% (v/v)
1- propanol as organic modifier in the ratio 70: 30 v/v and ultraviolet detection at 225 nm are used for the determination. Under
these conditions, the studied Aspirin and Atenolol elute between 4.033 ± 0.02 and 6.042 ± 0.031 min at a 1 mL/min flow rate.
Parameters such as linearity, precision, accuracy, specificity and robustness are studied as reported in the ICH guidelines. Linearity
for Atenolol and Aspirin was in the range of 2-12 μg/mL and 4-24 μg/mL respectively. The mean recoveries obtained for Atenolol
and Aspirin were 99.55 and 99.61 % respectively. Developed method was found to be accurate, precise, selective and rapid for
simultaneous estimation of Aspirin and Atenolol.
Keywords: Atenolol, Aspirin, MLC, simultaneous determination, validation.
INTRODUCTION
M
icellar liquid chromatography (MLC) is an
efficient alternative to conventional reversedphase liquid chromatography with hydroorganic mobile phases. Most MLC procedures uses hybrid
micellar mobile phases containing a surfactant above the
critical micellar concentration and a relatively small
amount of organic solvent to increase the elution
strength and improve the efficiencies. The anionic sodium
dodecyl sulphate (SDS) is the most usual surfactant in
MLC, but it also requires the addition of an organic
solvent to decrease the retention times and increase the
efficiency. In particular, positively charged basic
compounds are strongly retained by the stationary phase
modified by adsorption of SDS monomers and require the
addition of a strong solvent, such as propanol or
pentanol1-3.
Atenolol (ATN), 4-(2-hydroxy-3- [(1-methylethyl) amino]
propoxy] benzeneacetamide, is an antihypertensive,
antianginal, and antiarrhythmic4. Aspirin (ASP) is
chemically, 2-(acetyloxy) benzoic acid and exerts its antiinflammatory, analgesic and antipyretic actions. Aspirin
and other non-steroid anti-inflammatory drugs (NSAIDs)
inhibit the activity of the enzyme called cyclooxygenase
(COX) which leads to the formation of prostaglandins
5
(PGs) that cause inflammation, swelling, pain and fever .
Literature survey revealed that there are several methods
available to determine ATN and ASP either alone or in
combination with other drugs in pharmaceutical
formulations and biological fluids using various analytical
techniques such as spectrophotometric techniques6-8,
several methods based on separation techniques,
including HPTLC9-10, LC–MS11-12 and HPLC13-16 have been
also reported. There is no MLC method for the
simultaneous determination of these drugs in combined
dosage form. Hence a new MLC method has been
developed for the estimation of ATN and ASP in
combined dosage form. The developed method is simple,
precise, selective, and rapid and can be used for routine
analysis. The structures of the drugs are shown in Figure
1.
Figure 1a: Chemical structure of ATN
Figure 1b: Chemical structure of ASP
MATERIALS AND METHODS
Chemicals and Reagents
Aspirin and Atenolol were kindly supplied by Wockhardt
Pharmaceuticals Ltd., Aurangabad and Emcure
Pharmaceuticals Ltd, Pune, India. ATO-PRIN tablet
containing 50 mg Atenolol and 100 mg Aspirin were
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Int. J. Pharm. Sci. Rev. Res., 37(2), March – April 2016; Article No. 27, Pages: 151-155
obtained commercially within their shelf life. For MLC
work double distilled water was prepared in the
laboratory. 1-propanol, Sodium dodecyl sulphate,
disodium hydrogen phosphate and citric acid used were
of HPLC grade and analytical grade reagent and were
purchased from Merck Chemicals, Mumbai, India.
MLC instrument and chromatographic conditions
Chromatographic separation was achieved using MLC
System consisted of Intelligent HPLC pump model (Jasco
PU 2080 plus), an auto sampler and UV/VIS (Jasco UV
2075 plus) detector. The output signal was monitored and
processed using Jasco Borwin version 1.5, LC-Net II/ADC
software.
ODS Hypersil C18 (250 mm, 4.6 mm id, 5 µm particle size)
was used as the stationary phase. Mobile phase
consisting of 0.07M sodium dodecyl sulfate (SDS) pH 3
adjusted with phosphate buffer and 15 % (v/v) 1propanol as organic modifier in the ratio 70:30 v/v was
delivered at a flow rate of 1 mL/min. The detector was set
at the wavelength of 225 nm. Injection volume was kept
20 μL.
Preparation of standard and sample solutions
A standard mixed stock solution of atenolol and aspirin
was prepared by accurately weighing atenolol (50 mg)
and aspirin (100 mg) into a 25 mL volumetric flask. The
drugs were dissolved in methanol and the solution was
diluted to volume.
The stock solution was further diluted with mobile phase
to obtain a solution of ATN (2 μg/mL) and ASP (4 μg/mL),
respectively.
Twenty tablets of the pharmaceutical formulation ATOPRIN (containing 50 mg atenolol and 100 mg aspirin) were
assayed. They were crushed to a fine powder and an
amount of the powder corresponding to approximately
50 mg ATN and 100 mg ASP was weighed in a 25 mL
volumetric flask.
The powder obtained was dissolved in methanol. After
that, an adequate volume of aliquot was taken and
diluted with 0.07M SDS solution and sonication (for 30
min) the solution was diluted to volume with 0.07 M SDS
solution and filtered through 0.45 μm nylon membrane
filter (Pall India Pvt. Ltd). Finally, an aliquot of the clean
solution was injected into the chromatograph.
System suitability
From the filtered sample solution 2 μg/mL for ATN and 4
μg/mL for ASP were injected into the chromatograph. The
analysis was repeated six times to test the system
suitability for their retention time, resolution (Rs),
theoretical plates number (N) and tailing factors (T).
ISSN 0976 – 044X
range, LOD and LOQ, precision (intraday and interday
17
precision), accuracy and robustness .
Sample analysis
20 μL of working standard solution and sample solutions
were injected into the liquid chromatograph and the
chromatograms were recorded. From the peak area of
ASP and peak area of ATN the amount of the drugs in the
sample were calculated.
Linearity and range
For determining linearity, calibration curves were plotted
over a concentration range of 2-12 μg/mL for ATN and 424 μg/mL for ASP, respectively. A 20 μL of sample
solution was injected into the chromatographic system
using fixed volume loop injector. Chromatograms were
recorded. Calibration plots were constructed by plotting
peak area against the corresponding amount of each
drug.
Limit of detection and limit of quantitation
The LOD and LOQ were calculated according to the 3.3
σ/s and 10 σ/s criteria, respectively; where σ is the
standard deviation of the peak area and s is the slope of
the corresponding calibration curve.
Precision
Precision studies were carried out to establish the
intraday and interday precision of proposed method. The
intra-day precision (RSD, %) was assessed by analyzing
standard drug solutions within the calibration range,
three times on the same day. Inter-day precision (RSD %)
was assessed by analyzing drug solutions within the
calibration range on three different days over a period of
a week.
Accuracy
To check the accuracy of the developed method and to
study the interference of formulation additives, analytical
recovery experiments were carried out by standard
addition method, at 80, 100 and 120% level. The
experiment was conducted in triplicate. Percentage
recovery and relative standard deviation were calculated.
Robustness
To check the robustness of proposed method, small,
deliberate changes were made to the chromatographic
condition. A study was performed by changing the flow
rate and % of propanol in the mobile phase (± 1%).
Standard solution prepared as per test method and
injected into the MLC system. Flow rate change was done
by varying flow rate at from 0.9 mL/min to 1.1 mL/min.
RESULTS AND DISCUSSION
Method development
Method Validation
The developed MLC method was validated as per
International Conference on Harmonization (ICH)
guidelines for the parameters like specificity, linearity and
The MLC procedure was optimized for simultaneous
determination of ATN and ASP. Good resolution of both
the components was obtained with 0.07M sodium
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Int. J. Pharm. Sci. Rev. Res., 37(2), March – April 2016; Article No. 27, Pages: 151-155
dodecyl sulfate (SDS) pH 3 adjusted with phosphate
buffer and 15 % (v/v) 1-propanol as organic modifier in
the ratio 70:30 v/v.
for ASP, respectively. This indicates the method is
sufficiently sensitive.
The flow rate of 1 mL/min was optimum. UV detection
was made at 225 nm. At this wavelength ATN and ASP
can be quantified.
500000
Y = 26481*X + 108761
400000
A re a (A U )
Hence, 225 nm determined empirically has been found to
be optimum. The average retention times for ASP and
ATN was found to be 4.033 and 6.042 min, respectively
(Figure 2).
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0.9995
300000
200000
100000
0
0
5
10
15
C o n c e n tr a tio n µ g /m L
Figure 3: Calibration curve for ATN
500000
Y = 13211*X + 113353
0.9996
A re a (A U )
400000
Figure 2: Chromatogram of ASP and ATN
300000
200000
100000
System suitability
0
To ascertain its effectiveness, system suitability tests
were carried out on freshly prepared stock solutions. The
parameters obtained are shown in Table 1.
0
10
20
30
C o n c e n tr a tio n µ g /m L
Figure 4: Calibration curve for ASP
Table 1: System suitability parameters (n=6)
Parameters
ASP
ATN
Precision
Retention Time in min
4.033
6.042
Resolution (Rs)
0.00
4.729
Theoretical plates
number (N)
2959
3785
The precision of the method was expressed as relative
standard deviation (RSD, %). Results calculated as % RSD
values for intraday and interday precision studies are
shown in Table 2 and found to be satisfactory.
Tailing Factor
1.351
1.214
Accuracy
Linearity
Linearity was accessed by visualizing the calibration graph
and plot of the residuals.
The points distributed equally above and below the trend
line showed linearity.
The linear regression equations were Y = 26481X +
108761 (r2 = 0.9995) for ATN and Y = 13211X + 113353 (r2
= 0.9996) for ASP.
The plots obtained from linear regression are given in
Figures 3 and 4 for ATN and ASP, respectively.
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
Accuracy was determined at three levels 80%, 100% and
120% of the target concentration in triplicate and the
percentage recovery for the amount added was
calculated. The results are presented in Table 3.
Robustness
Robustness of the method was determined by performing
same analysis at slightly different parameters from the
optimized conditions of selected method. There were no
significant changes in the retention times of ASP and ATN
when the flow rate (± 0.1 mL/min.) and % of propanol in
the mobile phase (± 1%) were changed. The low values of
the % RSD indicate the robustness of the method, as
shown in Table 4.
The limits of detection and quantitation were found to be
0.8 μg/mL and 2 μg/mL for ATN and 2 μg/mL and 4 μg/mL
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Int. J. Pharm. Sci. Rev. Res., 37(2), March – April 2016; Article No. 27, Pages: 151-155
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Table 2: Precision studies
Conc.
(μg/mL)
Intra-day precision (n=3)
Measured Conc. ± SD
(%) RSD
Inter-day precision (n=3)
Recovery (%)
Measured Conc. ±SD
(%) RSD
Recovery (%)
3.973 ± 0.041
7.97 ± 0.090
11.91 ± 0.144
1.04
1.13
1.21
99.33
99.63
99.25
7.95 ± 0.083
15.92 ± 0.175
23.83 ± 0.27
1.05
1.10
1.14
99.38
99.50
99.29
Atenolol
4
8
12
3.975 ± 0.031
7.96 ± 0.072
11.92 ± 0.13
0.78
0.91
1.09
99.38
99.50
99.33
Aspirin
8
16
24
7.97 ± 0.089
15.96 ± 0.171
23.89 ± 0.244
1.12
1.07
1.02
99.63
99.75
99.54
Table 3: Recovery studies
Label claim
(mg/tablet)
Amount
Added (%)
Total amount (mg)
Amount recovered
(mg)
(%) Recovery
Mean (%) Recovery (± SD)
ATN
50
80
100
120
90
100
110
89.70
99.34
109.60
99.67
99.34
99.64
99.55 ± 0.182
ASP
100
80
100
120
180
200
220
179.44
198.83
219.37
99.69
99.42
99.71
99.61 ± 0.161
Table 4: Robustness evaluation of ATN and ASP
ATN
Conditions
Level
Rt (min.)
ASP
% RSD of peak area
Rt (min.)
% RSD of peak
area
A: Flow rate mL/min.
0.9
-0.1
6.095
1.02
4.092
1.11
1.0
0.0
6.042
1.10
4.033
1.05
1.1
+0.1
6.000
1.13
4.010
1.14
6.046 ± 0.048
1.083 ± 0.0569
4.045 ± 0.042
1.10 ± 0.0458
Mean ± SD
B: % of propanol in the mobile phase (± 1%)
% 14
-1.0
6.092
1.07
4.095
1.03
% 15
0.0
6.042
1.14
4.033
1.11
% 16
+1.0
6.004
1.19
4.012
1.18
6.046 ± 0.044
1.133 ± 0.0603
4.047 ± 0.043
1.107 ± 0.075
Mean ± SD
Analysis of marketed formulation
The chromatogram of the sample extracted from
conventional tablets showed peaks of ASP (Rt 4.033 min.)
and ATN (Rt -6.042 min) well resolved from other tablet
excipients. The percent contents of ASP and ATN per
tablet by proposed method was found to be 99.67 % and
99.60 %, respectively.
CONCLUSION
An accurate, sensitive and precise micellar liquid
chromatographic method has been developed and fully
validated for quality control analysis of atenolol and
aspirin in bulk and Pharmaceutical dosage form. The
developed method was found to be simple and have
certain advantages associated with this method such as
high selectivity, sensitivity and less hazardous. Moreover,
the lower solvent consumption along with the short
analytical run time leads to a cost effective and ecofriendly chromatographic procedure.
Acknowledgement: The authors would like to thank,
Emcure Pharmaceutical Ltd. Pune and Wockhardt
Pharmaceuticals Ltd., Aurangabad, India for providing a
gift sample of standard atenolol and Aspirin. The authors
would like to thank, Dr K R Mahadik, Principal, Poona
College of Pharmacy, Pune, India for providing necessary
facilities to carry out the work.
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Source of Support: Nil, Conflict of Interest: None.
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